Identification of protein phosphatase 4 catalytic subunit as a Wnt promoting factor in pan-cancer and Xenopus early embryogenesis

Protein Phosphatase 4 Catalytic Subunit (PPP4C) is an evolutionarily conserved protein involved in multiple biological and pathological events, including embryogenesis, organogenesis, cellular homeostasis, and oncogenesis. However, the detailed mechanisms underlying these processes remain largely unknown. Thus, we investigated the potential correlation between PPP4C and biological processes (BPs) and canonical Wnt signaling using pan-cancer analysis and Xenopus laevis (X. laevis) embryo model. Our results indicate that PPP4C is a potential biomarker for specific cancer types due to its high diagnostic accuracy and significant prognostic correlation. Furthermore, in multiple cancer types, PPP4C-related differentially expressed genes (DEGs) were significantly enriched in pattern specification, morphogenesis, and canonical Wnt activation. Consistently, perturbation of Ppp4c in X. laevis embryos interfered with normal embryogenesis and canonical Wnt responses. Moreover, biochemical analysis of X. laevis embryos demonstrated that both endogenous and exogenous Ppp4c negatively regulated AXIN1 (Wnt inhibitor) abundance. This study provides novel insights into PPP4C roles in pattern specification and Wnt activation. The similarities in BPs and Wnt signaling regulation regarding PPP4C support the intrinsic link between tumorigenesis and early embryogenesis.


Results
Highly conserved PPP4C is expressed ubiquitously among vertebrates. Amino acid (AA) sequence alignment in Fig. 1a reveals that PPP4C is highly conserved among vertebrate species. Specifically, X. laevis Ppp4c exhibits 99% identity with PPP4C in human (NP_001290432.1), mouse (NP_001347393.1), and rat (NP_599186.1) species. The expression pattern of PPP4C was explored in adult human and X. laevis transcriptome profiles and X. laevis embryos. Human single-cell and tissue RNA-seq analyses from The Human Protein Atlas (HPA) and GTEx showed that PPP4C was detected in all cell types and tissues, with higher transcriptional levels in reproductive, blood, and skin systems, and lower in muscle, brain, heart, and pancreas (Fig. 1b, Fig. S1). Likewise, X. laevis ppp4c were highly transcribed in the reproductive systems and lowly in muscle, pancreas, heart, and brain tissues (Fig. 1c), indicating its transcriptional conservatism among vertebrate species. X. laevis embryonic ppp4c expression was explored by RT-PCR, western blot, and whole-mount in situ hybridization (WMISH). Both the RNA and protein of ppp4c were ubiquitously detected in all embryonic stages (Fig. 1d,e). Notably, Ppp4c protein was slightly elevated between late gastrula and mid neurula stages (Fig. 1e). WMISH in Fig. 1f presents the ppp4c spatial information. Maternal ppp4c transcripts were localized in the animal hemisphere during cleavage (stage 3) and blastula (stage 8) stages. During gastrula stages (stage 10 and 11.5), the ppp4c-positive region extended to the dorsal and ventral marginal zone. In the neurula stage (stage 15), ppp4c was detected in the anterior part of neural plate and the neural plate border in the trunk region. As development proceeded to the tailbud stage (stage 32), ppp4c was enriched in the head region but almost undetectable in others. The PPP4C expression differences observed among different adult tissues and between adult and embryonic stages, together with its structural conservatism, promoted us to question its potential roles in embryogenesis and oncogenesis.
The transcriptional differences between tumor and normal tissues were further supported by the diagnostic index factor receiver operating characteristic (ROC) curve. ROC results in Table 1   Next, the prognostic potential of PPP4C was evaluated by the Kaplan-Meier (KM) estimate in TCGA. The transcription levels of PPP4C significantly correlated with patient OS in 15 tumor types, of which PPP4C was a risk factor for poor prognosis in ACC, BRCA, HNSC, KIRC, KIRP, LAML, LGG, LIHC, LUAD, MESO, PAAD, and UVM, whereas a protective factor for better OS in CESC, READ, and THYM (Fig. S3). Altogether, the distinct expression pattern between normal and tumor tissues, and the close correlation between PPP4C and OS indicate that PPP4C is a potential biomarker for tumorigenesis.

PPP4C-related DEGs are involved in multiple BPs.
To uncover PPP4C-related BPs in tumorigenesis, we analyzed the DEGs between PPP4C high (50%-100%) and low (0%-50%) expression tissues in each tumor. The criteria and numbers of DEGs selected for GO analysis in each tumor are listed in Table S1. The volcano plot of DEGs in LGG is shown in Fig. 2a. Interestingly, besides immune or DNA damage responses that facilitate or impede oncogenesis, PPP4C-related BPs focused on regionalization, pattern specification, morphogenesis, metabolism, and the development of skin, keratinocyte, epidermis, and muscle tissues (Fig. 2b,c). Notably, in BLCA, BRCA, CHOL, COAD, ESCA, LGG, MESO, PCPG, PRAD, SARC, THCA, and UCES, elevation of PPP4C correlated with activation of genes involved in the skin, epidermis, or keratinocyte-related processes (Fig. 2b PPP4C correlates with canonical Wnt signaling in tumors and X. laevis embryos. Given the fundamental function of Wnt in AP and DV pattern specification, and the correlation between PPP4C and pattern specification in certain tumor types, we explored the relationship between PPP4C and canonical Wnt activation Furthermore, we validated the correlation between Ppp4c and pattern specification, morphogenesis, and canonical Wnt signaling in the X. laevis embryo model, a fast and easy in vitro culture system. In this model, activation of canonical Wnt signaling in the ventral vegetal region or nearly totipotent animal cap (AC) region by ectopic β-catenin induced axis duplication or target gene transcription, respectively ( Fig. 3c-g). KD of Ppp4c in X. laevis embryos was achieved through injection of ppp4c morpholino (ppp4c MO), which was designed to block ppp4c translation by binding to its 5 ′ untranslated region (5 ′ UTR). The effectiveness and specificity of MO were confirmed by its ability to inhibit the translation of 5 ′ UTR containing ppp4c-ha (5 ′ UTR-ppp4c-ha) rather than MO-resistant non-5 ′ UTR containing ppp4c-ha (ppp4c-ha or Re.ppp4c-ha) (Fig. S4a,b). In Ppp4c-deficient embryos, the Wnt responses induced by wild-type β-catenin (WT-β-catenin or WT-BC) were compromised but could be rescued by co-injection of Re.ppp4c ( Fig. 3e-g).
To further identify which hierarchy level in Wnt signaling was affected by Ppp4c, we compared the Wnt responses induced by WT-β-catenin with those induced by constitutively active β-catenin (CA-β-catenin) in X.   www.nature.com/scientificreports/ laevis embryos. CA-β-catenin (S37A mutant) cannot be fully phosphorylated by GSK-3β and is therefore less susceptible to proteasome degradation 24 . The titration of CA-β-catenin to a comparable WT-β-catenin dose in axis duplication is shown in Fig. S5. Unlike WT-β-catenin-induced axis duplication and nodal3.1 transcription, which were reversibly inhibited upon loss of Ppp4c (Fig. 3f,g), both responses induced by CA-β-catenin were not affected by Ppp4c perturbation (Fig. 3h,i). Collectively, our pan-cancer data determined a correlation between PPP4C and canonical Wnt activation. In the X. laevis embryo model, Ppp4c promoted canonical Wnt responses at or upstream of β-catenin stability regulation.

X. laevis Ppp4c interacts with AXIN1 in vivo.
Co-immunoprecipitation (co-IP) assay of Ppp4c-HA with β-catenin and AXIN1 supported previous observations in X. laevis embryos. As shown in Fig. 4a, immunoprecipitates of Ppp4c-HA with an anti-HA antibody retrieved Myc-AXIN1, but not β-catenin-Myc in X. laevis embryo animal cap tissue. Consistently, we were able to detect Ppp4c-HA in the reciprocal immunoprecipitates of Myc-AXIN1. As the association between Ppp4c-HA and β-catenin was undetectable, we questioned whether Ppp4c regulated endogenous β-catenin through destruction complex. In the presence of Myc-AXIN1, endogenous β-catenin was detected in the immunoprecipitates of Ppp4c-HA (Fig. 4b), suggesting that Ppp4c indirectly associates with β-catenin through AXIN1. Moreover, co-IP of Ppp4c-HA with several truncated Myc-AXIN1 mutants showed that Ppp4c-HA interacted with several AXIN1 mutants lacking N-terminal parts, rather than the ones lacking C-terminal halves (Fig. 4c-e), suggesting that Ppp4c interacts with AXIN1 through the C-terminal halves.
X. laevis Ppp4c antagonizes AXIN1 abundance. Considering that the phosphorylation status of AXIN1 is closely associated with its stability and scaffold function, we postulate that Ppp4c may regulate AXIN1 abundance or scaffold function through dephosphorylation. As the endogenous Axin1 abundance in X. laevis embryos is below the western blot detection threshold, we evaluated ectopic Myc-AXIN1 regarding protein abundance and its interaction with endogenous β-catenin in embryos. In the lysate of stage 11 embryos, ectopic Myc-AXIN1 abundance was dose-dependently decreased by co-injection of ppp4c-ha mRNA (Fig. 5a), and reversibly accumulated in Ppp4c-deficient embryos (Fig. 5b,c), conveying that Ppp4c inhibits AXIN1 at the protein level. Furthermore, as ectopic Myc-AXIN1 in Ppp4c-KD embryos shifted behind the normal ones (Fig. 5c, lanes 2 and 3), and two catalytic-dead Ppp4c mutants (D82A, H115Q) failed to inhibit Myc-AXIN1 abundance (Fig. 5d, lanes 1-5), AXIN1 inhibition is most likely to be correlated with its protein modification, such as dephosphorylation. Notably, Ppp4c could partially rescue AXIN1 band shift in the presence of Wnt8 (Fig. 5c, Lane 2-4 and 7), suggesting that exogenous Wnt ligand promotes Ppp4c's function in AXIN1 protein modification. Additionally, we observed that the tail motif deletion Ppp4c mutant ( DYFL) expressed enhanced Myc-AXIN1 inhibition compared to WT-Ppp4c (Fig. 5d, lanes 2, 3, and 6), implying that the tail motif may negatively regulate Ppp4c activity in AXIN inhibition during gastrula stages. In the scaffold function study, to normalize precipitated AXIN1 abundance within groups, we utilized a TANKs domain truncated AXIN1 mutant (Myc-AXIN1-C1), which possessed interaction sites with both β-catenin and Ppp4c (Fig. 4c,d), and its abundance was not Ppp4c-dependent (Fig. 5e,f). Perturbation of Ppp4c rarely affected the abundance of endogenous β-catenin that immunoprecipitated with Myc-AXIN1-C1 (Fig. 5f,g). Collectively, these observations indicate that Ppp4c inhibits AXIN1 abundance in a catalytic-dependent manner.
Perturbation of Ppp4c interferes with normal embryogenesis in X. laevis. In pan-cancer study, PPP4C levels correlated with multiple BPs, such as regionalization, pattern specification, morphogenesis, metabolism, and the development of skin, keratinocyte, epidermis, and muscle tissues. In both X. laevis embryos and certain tumor tissues, PPP4C levels significantly correlated with canonical Wnt activation. Therefore, we questioned whether interference with Ppp4c could affect normal embryogenesis. As activation of canonical Wnt signaling in dorsal animal (DA) or ventral vegetal regions of embryos could induce head truncation or axis duplication respectively, we wondered whether excess Ppp4c could generate similar phenotypes. Overexpression of Ppp4c in the DA region, where the future head structure originates from, dose-dependently reduced the eye field, cement gland, and overall head structures in tailbud embryos (Fig. 6a,b). In addition, WMISH of several markers of head structure shows that Ppp4c DA overexpression reduced bf1 (forebrain) and pax6 (eye), rather than krox20 (hindbrain) (Fig. 6c), indicating excess Ppp4c alters AP patterning in embryogenesis. Unlike β-catenin (50 pg), injection of ppp4c or ppp4c-DYFL (up to 2 ng) failed to induce axis duplication when injected ventral-vegetally (Fig. S6a), implying that Ppp4c enhances rather than directly induces canonical Wnt activation in X. laevis embryos and may require the cooperation from other Wnt axis levels.
In KD studies, we first validated the efficiency of injection sites, and the most efficient and consistent KD phenotype was observed in 1-cell stage AC injected embryo (Fig. S6b). Injection of ppp4c MO into animal hemisphere of 1-cell stage embryos dose-dependently impaired anterior structures and melanocytes located on dorsal and lateral regions (Fig. 6d,e). The head size, retinal pigment epithelium (RPE), and melanocyte defects in Ppp4c-KD embryos can be partially rescued by co-injection of Re.ppp4c mRNA (Fig. 6f,g), suggesting that Ppp4c is essential for proper development of anterior structures and melanocytes during X. laevis embryogenesis. Notably, anterior structure defects in Ppp4c-KD embryos differ from those in Ppp4c overexpression embryos. In Ppp4c-KD embryos, the cement gland size was unaffected and anterior structures were not fully eliminated, indicating that the anterior deficiencies in the Ppp4c-KD embryos are more likely caused by tissue-specific rather than global AP patterning defects. Collectively, perturbation of Ppp4c in early embryos interferes with normal embryogenesis.

Discussion
Like other serine/threonine phosphatases, PP4 participates in multiple cellular processes, including DNA damage response, cell cycle regulation, tumorigenesis, immune response, stem cell pluripotency maintenance, and differentiation 11,[25][26][27][28][29] . In human cancer RNA-seq profiles and X. laevis embryos, we explored the function of PPP4C, the PP4 catalytic subunit. PPP4C levels correlated with pattern specification and canonical Wnt activation in both fields. Specifically, we propose that Ppp4c promotes canonical Wnt activity at the destruction complex level, as X. laevis Ppp4c interacted with AXIN1 and catalytically inhibited its protein abundance. Furthermore,   www.nature.com/scientificreports/ Perturbation of certain genes which are active in embryos and silent in adult tissue can tip the balance between proliferation and differentiation, inducing congenital diseases or cancer. Scattered evidence of PPP4C elevation has been documented in breast, lung, ovarian, colorectal, and pancreatic ductal tumors [10][11][12][13][14] . In lung cancer and colorectal carcinoma, PPP4C promoted tumorigenesis by facilitating tumor cell survival, proliferation, migration, and invasion 10,13 . In ovarian cancer cells, PP4 inhibition by fostriecin or KD enhanced immune response and reduced homologous recombination in tumor cells, leading to tumor repression 14 . In breast tumor cells, PPP4C can act as either an anti-tumor factor promoting cell apoptosis through PEA15 regulation or an oncogenic factor facilitating proliferation and migration in a cell-content dependent manner 9,30 . However, to our knowledge, no research has addressed its diagnostic or prognostic potential in pan-cancer. Consistent with previous reports, PPP4C mRNA levels were significantly upregulated in most cancers (27/31) and decreased in LAML. Moreover, the unbalanced transcriptional levels of PPP4C between normal and tumor tissues contributed to its high diagnostic accuracy in 14 tumor types. In tumorigenesis, PPP4C probably behaves as an oncogenic or protective factor cell-content-dependently, as its transcription levels corresponded to different OS outcomes in various cancer types. PPP4C-related DEGs in specific tumors were also consistent with the existing roles of PPP4C, as the DEGs closely correlated with immune response and DNA replication-related processes.
Additionally, PPP4C levels in multiple cancers were closely associated with various BPs, including morphogenesis, regionalization, and pattern specification. The DV patterning role of Ppp4c has been reported in zebrafish model. Depletion of Ppp4ca or Ppp4cb resulted in embryo dorsalization, as indicated by the expansion of organizer-specific marker chordin, and reduction of ventral marker gata2 at the shield stage 8 . However, injection of ppp4c MO into the future dorsal marginal zone of 4-cell stage X. laevis embryos, rarely affected the dorsal organizer (gsc) domain at the gastrula stage (data not shown). As the ppp4c domain was not spatially overlapped with the organizer, we postulated that zygotic Ppp4c is not essential for organizer formation. However, due to MO limitations, we cannot preclude that maternally inherited Ppp4c protein participates in organizer shaping processes. In X. laevis model, Ppp4c correlated with AP pattern specification, as overexpression of Ppp4c in DA dose-dependently reduced anterior structures at the tailbud stage. Reduction of forebrain (bf1) and eye (pax6) markers in neurula embryos, without impact on hindbrain marker (krox20), further corroborated its anteriorinhibition effect. The discrepancies between zebrafish and X. laevis models may be due to: (1) other phosphatases functional redundancy; (2) protein modifications affecting PP4 complex assembly; and (3) zebrafish and higher vertebrate PPP4C structural differences at N ′ terminal 9 AA, which may lead to binding regulatory subunits and further substrates differences. The developmental role of Ppp4c in anterior structures and melanocytes was supported by the KD studies, and the ppp4c positive domain overlapped with neural crest tissue, which has potential to develop into future sensory organs, melanocytes, ganglia, and facial skeleton structures. Interestingly, DEGs in GBM, UVM, and LAML were significantly enriched in retina, melanosome, or pigment-related BPs (Supplementary Dataset File 4), further supporting the similarities between embryogenesis and tumorigenesis. We also observed that PPP4C-related DEGs in specific tumors were involved in epidermis, skin, keratinocyte, and muscle-related BPs. As PPP4C was transcribed unevenly between skin and muscle in both human and X. laevis tissue RNA-seq profiles, exploring the potential of PPP4C in epidermis/muscle cell fate determination in embryonic and adult tissue will benefit future studies. These cellular response similarities imply that the tumors mentioned above may share similar cell content with embryos concerning each biological process, and clues from cancer studies may provide alternative views for embryonic research. Therefore, X. laevis embryos may serve as an alternative model for cancer research and vice versa.
PPP4C is reported to regulate Wnt, BMP, and Hedgehog signaling pathways 8,22,31 . We specifically explored the relationship between PPP4C and Wnt signaling due to its fundamental function in AP patterning and neural crest formation. The positive genetic interaction between PP4 and Wnt signaling was initially described in Drosophila through an RNAi screen 32 . In further studies, PP4 enhanced Notch-driven wnt ligand wg transcription or initiated the nuclear translocation of the receptor-like tyrosine kinase (Ryk) intracellular domain to promote Wnt signaling 22,23 . A Wnt inhibition role was reported in embryonic stem cells rather than cancer cells. The PP4 complex promoted histone deacetylation, inhibiting Wnt target gene brachyury transcription 25 . In our study, PPP4C levels correlated with canonical Wnt activation in multiple cancers, and X. laevis Ppp4c was required for canonical Wnt responses in embryos. Overexpression of Ppp4c in the DA region reduced the most anterior structures, phenocopying weak Wnt activation in embryogenesis. Unlike neural crest-derived melanocytes necessitating persistent Wnt for specification, RPE pigment cells originate from the forebrain and require dynamic Wnt regulation. Although we did not observe apparent DV defects by ppp4c MO as seen in zebrafish, the BMP regulatory activity may also be required for Ppp4c-mediated anterior structure formation in X. laevis embryos. Therefore, the functional time-point, region, and the cooperation between its Wnt and BMP regulation in Ppp4cmediated RPE and melanocyte development, are yet to be determined in future studies.
AXIN abundance is several magnitudes lower than other components in the destruction complex, thus, regulation of AXIN abundance and scaffold function is essential for canonical Wnt signaling regulation. AXIN phosphorylation, by GSK3 and CK1, stabilizes it and enhances its binding affinity for β-catenin and GSK3 in the destruction complex [33][34][35] . In Wnt "ON" status, AXIN is dephosphorylated, leading to the dissociation of β-catenin from the destruction complex and subsequent AXIN degradation 36 . Several PP complexes (PP1, PP2A, and PP2C) have been reported to interact with AXIN. PP1 antagonizes CK1 on the phosphorylation status of AXIN, releasing it from GSK3 37 . Moreover, PP1 changes AXIN conformation into "inactivation" mode upon LRP6 phosphorylation, diminishing its binding to phospho-LRP6, leaving it unperturbed for continuous signaling 38 . PP2C decreases AXIN half-life, eliciting a synergistic response with β-catenin and Wnt1 in cells 36 . Another AXIN interactor, PP2A, contributes to Wnt inhibition possibly through dephosphorylation of GSK3β 39 . In this study, Ppp4c was identified as a Wnt-promoting factor in X. laevis. Unlike previously reported Wnt-promoting roles, Ppp4c was proposed to target AXIN1 in β-catenin regulation, which was supported by the following evidence: (1) unlike WT-β-catenin, stable CA-β-catenin-induced Wnt responses were Ppp4c perturbation-resistant; (2) Scientific Reports | (2023) 13:10240 | https://doi.org/10.1038/s41598-023-35719-y www.nature.com/scientificreports/ AXIN1, particularly the C-terminal halves, co-precipitated with Ppp4c; (3) β-catenin could not be detected in the immunoprecipitates of Ppp4c unless AXIN1 was present; and (4) X. laevis Ppp4c inhibited AXIN1 abundance in a catalytic-dependent manner. AXIN can be subjected to either PARsylation/ubiquitination by Tankyrase/RNF146 or ubiquitination by Smurf2 for proteasome degradation [40][41][42] . In our study, TANKs domain truncated AXIN1 mutant was resistant to Ppp4c perturbation, suggesting that PARsylation by Tankyrase may be involved in Ppp4c-mediated AXIN1 degradation. Interestingly, DYFL motif deletion in Ppp4c enhanced its AXIN1 inhibition in gastrula embryos. The tail motif, which is highly conserved in PP2A family catalytic subunit factors, is subject to methylation and phosphorylation modification [43][44][45] . For PP2AC, DYFL methylation or phosphorylation within or upstream was found to differentially affected subunit affinity, thereby tipping the balance between different PP2A complexes 43 . Likewise, demethylation in the PPP4C DYFL motif diminished its interaction with PP4R1, PP4R2, PP4R3α , and PP4R3β 45 . Considering that the substrate specificity is mainly determined by binding subunits and the DYFL motif modifications affect PP complex assemble, we postulate that DYFL modification during the gastrula stage is unfavorable for AXIN-inhibitory PP4 complex formation. Notably, Ppp4c or Ppp4c-DYFL failed to induce axis duplication when injected ventral-vegetally. Based on the existing literature and our data, we postulate that this may be due to three possibilities as below: (1) abundance limitation of the PP4 regulatory subunits or other co-factors. In ventral vegetal overexpression studies, these proteins may be the limiting factors for the formation of Wnt-promoting PP4 machinery. In this case, insufficient co-factors or PP4 complex may compromise the Wnt responses in the ventral vegetal region when excess Ppp4c present. (2) BMP inhibition. In C2C12 cells, PPP4C was found to interact with Smad1 without affecting its linker or C-terminal phosphorylation, and the nuclear translocation of p-Smad1 shuttled PPP4C from the cytosol to the nucleus 8 . Therefore, in the ventral region, the accumulation of p-Smad1 and its nuclear translocation upon BMP activation may favor the nuclear rather than cytosolic PP4 complex formation, reduce the interaction between Ppp4c and AXIN1, and ultimately affect the Wnt regulatory activity of Ppp4c. (3) Canonical Wnt activity requirements. Ppp4c could partially rescue AXIN1 band shift in KD embryos with exogenous Wnt8. However, endogenous Wnt activity is not completely blocked in X. laevis embryos, therefore, whether Wnt activation is required for Ppp4c-mediated AXIN1 inhibition, and if Ppp4c dephosphorylates other Wnt signaling components to degrade AXIN, are yet to be determined.
These findings collectively provide a novel Ppp4c function in regulating canonical Wnt signaling. However, this research had some limitations due to technical issues. In embryo model, as massive amounts of β-catenin exist on the plasma membrane, perturbation of cytosolic β-catenin, by loss or gain of Ppp4c, was not sensitively detected by western blot. Additionally, in our unpublished data, excess of Wnt8 enhanced the AXIN1 inhibitory activity of Ppp4c in Xenopus embryos. In future investigations, it would be fascinating to investigate the following: (1) whether Ppp4c promotes canonical Wnt signaling through endogenous Axin1 or not; (2) if so, whether this mechanism can be applied to the tumors identified in pan-cancer analysis; (3) the binding regulatory subunit(s) for Wnt activation and the PP4 assembly regulation; (4) if and how Ppp4c dephosphorylation correlates with Wnt/Tankyrase/RNF146-mediated AXIN degradation; and (5) whether Ppp4c regulates other Wnt axis levels and how these regulations interact. A detailed analysis will uncover the precise mechanism of Ppp4c in canonical Wnt signaling and the involvement of other pathways in this regulation.

Materials and methods
Data acquisition. The RNA-seq data of normal human tissues [GTEx Analysis V8 (dbGaP Accession phs000424.v8.p2)] and single cell were downloaded from GTEx (https:// www. gtexp ortal. org/ home/ datas ets) and HPA (https:// www. prote inatl as. org/ about/ downl oad) respectively. The expression feature of PPP4C in pancancer and its diagnostic value were explored in a combined cohort study of TCGA and GTEx downloaded from UCSC Xena (https:// xenab rowser. net/ datap ages/). The analysis of prognostic value, DEGs, GO enrichment, and GSEA were performed in the transcriptome data across 33 tumor types downloaded from the TCGA (https:// portal. gdc. cancer. gov) together with the relevant clinical profile.
Diagnostic and prognostic value analysis. The diagnostic potential of PPP4C in each cancer type was assessed by the ROC curve in the cohort of TCGA and GTEx data. The accuracy of prediction was presented by the area under the curve (AUC), which was calculated by the package pROC (v 1.18.0). AUC values were classified as high accuracy (> 0.9), certain accuracy (> 0.7 and ≤ 0.9), and low accuracy (> 0.5 and ≤ 0.7).
KM plots were used to investigate the association between PPP4C level and patients OS in pan-cancer. The cutoff value for PPP4C high and low expression groups was determined using the minimum p value method 46 . To avoid extreme imbalance in group numbers, the cutoff value in each cancer was restricted between the upper and lower quartile of PPP4C values. The survminer package (v 0.4.9) was used for visualization, and the survival package (v 3.4-0) was used for statistical analysis. p < 0.05 was considered as statistically significant.
GO enrichment analysis and GSEA of the DEGs. DEGs were analyzed using the DESeq2 package (v 1.36.0) based on the gene counts data between PPP4C high and low groups (divided by the median). The minimal values of |log2FoldChange| and numbers of DEGs (p.adjust < 0.05) in each tumor type selected for GO enrichment analysis are listed in Table S1. GO enrichment analysis of the DEGs was conducted using the clus-terProfiler package (v 4.4.4). In each tumor, the top 25 GO biological process items with a p.adjust value < 0.05 were collected for word cloud analysis using the wordcloud2 (v 0.2.1) and jiebaR (0.11) packages.
GSEA was performed on DEGs to determine the correlation between canonical Wnt activation and PPP4C level. The clusterProfiler package was used for statistical analysis, and the enrichplot package (v 1. 16 Embryos manipulation and microinjection. X. laevis eggs were obtained, fertilized, and staged as previously described 47,48 . Embryos were cultured in 0.33×Modified Ringer (MR) until the mid-blastula transition, after which transferred to 0.1× MR until harvest. Embryo injection was performed using HARVARD Injector in 0.33× MR containing 4% Ficoll-PM400 (GE Healthcare) at indicated stages. Injected embryos were cultured in 4% Ficoll-400 in 0.33× MR before the mid-blastula transition. For the axis duplication assay, mRNA or morpholino was injected into the ventral vegetal region at the 4-cell stage, and the embryos were cultured until the tailbud stage (Fig. 3c) 49 . In the animal cap explant induction experiment, animal caps were dissected at stage 8 and cultured until the sibling embryos reached stage 10.5 (Fig. 3d)  RT-PCR. The target gene abundance was analyzed semi-quantitatively by RT-PCR. 5 or 10 whole embryos or 30 explants were harvested as described and snapped in liquid nitrogen. Total RNA was extracted from the samples using TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. Purified total RNA (5 µ g) was reverse transcribed into cDNA using the M-MLV reverse transcriptase (Promega). odc1 was used as an internal control for normalization. Amplification of each target gene was performed in a 25 µ l system using Taq polymerase (Solgent). Primers used in RT-PCR are listed in Table S2.
WMISH and lacZ β-galactosidase Staining. WMISH was performed as previously described and stained with NBT/BCIP (Sigma) 51 . The antisense in situ probe labeled with digoxigenin (DIG) against ppp4c was transcribed using T7 RNA polymerase with the template generated by linearization of ppp4c-T-easy construct with SpeI. The probes against bf1, pax6, and krox20 were obtained as previously described 52,53 . Indication of injection site was performed by lacZ β-galactosidase staining. 250 pg of lacZ mRNA was coinjected into the embryos. β-galactosidase-expressing embryos at the indicated stage were fixed in MEMFA solution (10% formaldehyde, 0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO 4 ) for 30 min at room temperature after being rinsed with PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ). Then, the embryos were stained for 1-2 h at room temperature in PBS containing 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 1 mg/ml X-gal (red), 2 mM MgCl 2 . The stained embryos were fixed in MEMFA solution for 4 h for further WMISH.
Western blot, immunoprecipitation, and antibodies. Western blot assays and co-IP were carried out as previously described 49,54 . Briefly, embryos or animal cap explants were homogenized in Triton-X 100 lysis buffer [20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 140 mM NaCl, 10% glycerol, 1 mM EGTA, 1.5 mM MgCl 2 , 1 mM DTT, 1 mM sodium vanadate ( Na 3 VO 4 ), 50 mM NaF, 20 µg/ml aprotinin, 40 µg/ml leupeptin, and 0.75 mM phenylmethylsulfonyl fluoride (PMSF)] for western blot assay, or IP buffer [20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 2 mM EDTA (pH 7.5), 1% TritonX-100, 1 mM Na 3 VO 4 , 100 mM NaF, 25 mM β-glycerophosphate, 20 µg/ml aprotinin, 40 µg/ml leupeptin, and 0.75 mM PMSF] for co-IP assay. The lysate was centrifuged to remove insoluble debris and yolk. In western blot, an equivalent amount of 1/4 whole embryo protein was loaded for each well of the SDS-PAGE gel, and β-Actin was used as the internal control. In co-IP assay, the indicated antibodies were added to the supernatants and incubated at 4 • C for 6 h. Rec-Protein G-Sepharose 4B Conjugate (Invitrogen) was added, and the mixture was incubated at 4 • C overnight. The immune complexes bound to Protein-G beads were washed six times with IP buffer and then subjected to SDS-PAGE gel for western blot. The mobility shift assay was performed on a 6.5% SDS-PAGE gel. The antibodies used in western blot and co-IP were listed in Table S3. Immunoreactivity was visualized by incubation with SuperSignal TM West Femto Maximum Sensitivity Substrate (Thermo) following the manufacturer's instructions or with ECL solution unless specified. For the blots in western blot that required equal or more than 3 antibodies hybridization, and co-IP experiments, the membranes were cropped at indicated region before antibody hybridization to reduce bleaching times and non-specific staining interference .
Statistical analyses and image process. Statistical analysis was conducted using R software (v 4.2.1) downloaded from R website (https:// www.r-proje ct. org/). The Wilcoxon rank sum test was used to compare the difference of PPP4C between normal and tumor tissues in non-paired and paired samples. For the phenotype and western blot analysis, the comparison between groups was assessed by Student's t-test. The p value was calculated using the rstatix package (v 0.7.0). The ggplot2 package (v 3.3.6) was used for visualization unless specified. p < 0.05 was considered statistically significant (ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001). PPP4C